Bird Louise E, Chamberlain Philip P, Stewart-Jones Guillaume B E, Ren Jingshan, Stuart David I, Stammers David K
Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, Henry Wellcome Building of Genomic Medicine, University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7BN, UK.
Protein Expr Purif. 2003 Jan;27(1):12-8. doi: 10.1016/s1046-5928(02)00567-3.
A purification procedure is described for the isolation of recombinant HIV-2 reverse transcriptase expressed in Escherichia coli. The p68 subunit is expressed, in the absence of induction, and use of a heparin-Sepharose column produces substantially pure protein. Concentration of the homodimeric p68 reverse transcriptase pool, followed by incubation at room temperature for several days, results in full conversion by E. coli proteases to the heterodimer (p68/p55). This extended incubation simplifies the purification process and improves the yield of heterodimeric reverse transcriptase, which shows a truncation of the smaller subunit to 427 residues. The protein is then purified further by hydroxyapatite and gel-filtration chromatography to homogeneity. The HIV-2 RT is active and has been used to produce crystals that diffract to beyond 3.0 A.
本文描述了一种用于分离在大肠杆菌中表达的重组HIV-2逆转录酶的纯化方法。在未诱导的情况下表达p68亚基，使用肝素-琼脂糖柱可产生基本上纯的蛋白质。对同二聚体p68逆转录酶池进行浓缩，然后在室温下孵育数天，大肠杆菌蛋白酶可将其完全转化为异二聚体（p68/p55）。这种延长的孵育简化了纯化过程，并提高了异二聚体逆转录酶的产量，该异二聚体显示较小亚基截短至427个残基。然后通过羟基磷灰石和凝胶过滤色谱进一步纯化该蛋白质至均一性。HIV-2逆转录酶具有活性，并已用于产生衍射分辨率超过3.0埃的晶体。
